Immunotherapies directed against currently defined tumor-associated or tumor-specific antigens can enhance anti-tumor immunity in patients, as detected with in vitro immune monitoring, and yet have had limited clinical success (1). One reason may be the nature of the targeted antigens, the majority of which are proteins overexpressed in tumor cells but not essential to maintaining their malignant phenotype.
Phosphorylation is the most common and ubiquitous form of enzyme-mediated post-translational protein modification, and transient phosphorylation of intracellular signaling molecules regulates cellular activation and proliferation (2). Phosphorylation cascades are often dysregulated during malignant transformation, leading to uncontrolled proliferation, invasion of normal tissues, and distant metastasis (3, 4). Limited but growing evidence has shown that tumor-associated phosphoproteins processed intracellularly through an endogenous pathway can give rise to phosphopeptides complexed to MHC I molecules which are displayed on the cell surface (5, 6). CD8+ T cells immunized to specifically recognize these phosphopeptides are also capable of recognizing intact human tumor cells, suggesting that phosphopeptides may represent a new class of targets for cancer immunotherapy (5, 6) (ALZ and VHE, unpublished data). In these studies and others, T cell discrimination of the phosphopeptide versus its non-phosphorylated counterpart was observed, indicating that phosphorylation can influence peptide immunogenicity (5-13). Recent crystal structural definition of phosphorylated peptide-HLA-A2 complexes demonstrated direct and indirect interactions of the phosphoresidue with the MHC molecule, often significantly increasing the affinity of the phosphopeptide for MHC I. Additionally, phosphoresidues were solvent-exposed, suggesting the potential for direct interactions with the T cell receptor (TCR) (14, 15).
Mounting evidence indicates that MHC II-restricted CD4+ T lymphocytes are a critical component of anti-tumor immunity, and that their activation and recruitment may be required to optimize cancer immunotherapies (16, 17). A variety of post-translational modifications have been identified on naturally processed MHC class II-associated epitopes. These include N- and O-linked glycosylation, N-terminal acetylation, nitration, deamidation, and deimination/citrullination (18). Although an early attempt to detect phosphorylation on class II MHC peptides met with failure (5), new technology has now made it possible to observe this modification as well (19). Here we demonstrate the existence of MHC II-associated phosphopeptides on human melanoma cells and EBV-transformed B (EBV-B) lymphoblasts, and we define and compare the sequences of phosphopeptides complexed to HLA-DR molecules on two autologous pairs of melanoma and B cell cultures. Furthermore, we show for the first time the ability of human CD4+ T cells to specifically recognize phosphoepitopes displayed in the context of MHC II molecules, using the example of an HLA-DRβ1*0101-restricted phospho-MART-1 peptide isolated independently from two melanoma cell lines. These findings suggest that tumor-associated phosphopeptides provide targets for CD4+ as well as CD8+ T cells, potentially enabling the development of new immunotherapeutic strategies.
There is a continuing need in the art to identify new therapeutic targets and new markers of disease.